Pair of measuring electrodes, biosensor comprising a pair of measuring electrodes of this type, and production process

ABSTRACT

A pair of measuring electrodes comprising a first and a second, preferably in each case sheet-like electrode comprises an insulation layer arranged between said electrodes. One or more nanopores, which extend through said insulation layer as far as said first electrode, the surface of which is at least partially uncovered by said nanopores, are provided in each second electrode. The invention also describes a biosensor comprising a pair of measuring electrodes of this type, an electrochemical cell comprising a biosensor of this type and a process for producing said pair of measuring electrodes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/360,793, entitled PAIR OF MEASURING ELECTRODES, BIOSENSOR COMPRISINGA PAIR OF MEASURING ELECTRODES OF THIS TYPE, AND PRODUCTION PROCESS,filed Jan. 27, 2009, which is a continuation of U.S. application Ser.No. 10/688,771, entitled PAIR OF MEASURING ELECTRODES, BIOSENSORCOMPRISING A PAIR OF MEASURING ELECTRODES OF THIS TYPE, AND PRODUCTIONPROCESS, filed Oct. 16, 2003, which is a continuation of InternationalPatent Application PCT/EP02/04222 filed on Apr. 16, 2002, anddesignating the US, which was not published under PCT Article 21(2) inEnglish, and claims priority of German Patent Application DE 101 20083.8, filed on Apr. 17, 2001, which is incorporated herein byreference.

BACKGROUND

1. Field

The field relates to a pair of measuring electrodes, to a biosensorcomprising at least one pair of measuring electrodes of this type, to anelectrochemical cell comprising a biosensor of this type and to aprocess for producing the pair of measuring electrodes.

2. Description of the Related Prior Technology

Numerous pairs of measuring electrodes and biosensors of this type areknown from the prior art; cf. for example WO 99/07879, WO 00/62047 or WO00/62048, as well as the very wide range of possible applications anduses described therein.

The biosensors described in these documents have arrays of pairs ofmeasuring electrodes which can be addressed individually and each ofwhich pairs has two electrodes which are arranged parallel to oneanother in one plane and may be designed as fingers entangled into oneanother, so-called interdigitated electrodes, or as interleaved,concentric sections of a circle.

The known biosensors are used, for example, to determine concentrationsof biomolecules, to determine physico-chemical properties, to detectimmune reactions and the like. In general terms, biosensors of this typeare used in analytical or immunological assays, where they are used asamperometric sensors to measure extremely low concentrations. However,the measuring electrodes can also be used for electrostimulation, forelectrophoretic enrichment or separation of charged molecules or, forexample, for electrochemical recording of reaction sequences.

The amperometric sensors mentioned detect currents which emanate fromoxidation or reduction reactions at molecules in solution in thevicinity of the electrodes. A selectivity for a specific moleculespecies can be achieved because certain redox-active molecules arereduced or oxidized at a specific potential. The current measured isproportional to the concentration of the molecules in the solution.

Depending on the type of reaction involved, the transferred charge permolecule is one or a few elemental charges, whereby it is possible toincrease the sensitivity by what is known as redox recycling. For thispurpose, the electrodes are arranged at a very short distance from oneanother, so that a redox-active molecule can diffuse to and fro betweenthe anode for the oxidation reaction and the cathode for the reductionreaction with a high probability. In the process, the molecule takes upcharge a number of times at the cathode (reduction) and releases itagain at the other electrode, the anode (oxidation); cf. Niwa et al.,Electroanalysis 3 (1991), 163-168.

To enable the redox recycling phenomenon to be exploited, the dimensionsof and distance between the electrodes must be as small as possible inorder to allow a rapid diffusion of the molecules between anode andcathode. Niwa et al., loc. cit., in this context describe two differentelectrode arrangements, in which the distances between anode and cathodeand the widths of anode and cathode are as little as 1 μm and the lengthis 2 mm. In one embodiment, up to 100 interdigitated fingers lie next toone another in one plane, while in the other embodiment anode andcathode fingers are vertically spaced apart by an insulation, resultingin a regularly structured array having microstrips. The electrodes areproduced by conventional photolithography and etching techniques.

In order to achieve the selectivity, which is not sufficient in the caseof the vertical arrangement, the authors propose a further reduction inthe dimensions or the use of selected polymers.

SUMMARY OF CERTAIN INVENTIVE ASPECTS

Against this background, one object underlying the present invention isto provide a pair of measuring electrodes of the type mentioned at theoutset which has a very high sensitivity and is simple and inexpensiveto produce.

According to the invention, this object is achieved by a pair ofmeasuring electrodes comprising a first and a second, preferably in eachcase planar or sheet-like electrode and an insulation layer arrangedbetween the electrodes, in which pair one or more nanopores are providedin the second electrode, extending through the insulation layer to thefirst electrode, the surface of which is at least partially uncovered inthe nanopores.

This object underlying the invention is thereby completely achieved.

This is because the inventor of the present application has recognizedthat it is possible to build up the pairs of measuring electrodes fromin each case two, preferably sheet-like electrodes which are arrangedparallel and are spaced apart from one another by an insulation layer,and to provide nanopores in the upper electrode, which nanopores extendthrough the insulation layer down to the lower electrode, which lowerelectrode is therefore partially exposed in the nanopores. In thecontext of the present invention, the term “nanopores” is understood asmeaning openings or recesses which in cross section are not necessarilycircular or of any other regular shape and which have an opening widththat is submicron. The width may be, for example, from approximately 20to 500 nm. In one embodiment it may be approx. 100 nm. In certainembodiments, the depth of the nanopores is substantially determined bythe thickness of the insulation layer, which amounts to approximately 10to 200 nm. In one embodiment, the thickness of the insulation layer maybe approx. 50 nm.

The nanopores do not have to be arranged regularly, but rather they maybe arranged irregularly or randomly regularly. The number of nanoporesper unit area, i.e. their density, may vary within a wide range,provided that it is ensured that the perforated upper electrode stillremains conductive in the lateral direction and that adjacent nanoporesare separated from one another by remaining material of the insulationlayer.

The inventor has realized that, by turning away from the standard fingerelectrodes, it is possible to create pairs of measuring electrodes, inparticular for biosensors, which have a very high sensitivity andselectivity, since not only the distance between the electrodes can bereduced virtually to any desired degree, but also the large number ofnanopores which is possible per pair of measuring electrodes also meansthat an extremely high number of molecules contributes to themeasurement current in the redox recycling process. On account of thesmall diameter of the nanopores and the small thickness of theinsulation layer, there are also very short diffusion paths, meaningthat the molecules can take up charge and release it again veryfrequently per unit time, which likewise contributes to increasing thesensitivity.

In this context, it is preferred if the electrodes have a diameter ofapprox. 1 μm to approx. 10 mm, preferably of approx. 10 μm, the pair ofmeasuring electrodes preferably being applied to an insulating substratewhich further preferably comprises glass, silicon/silicon oxide or apolymer. The electrodes may in this case contain metal, preferably gold,platinum, palladium, iridium or carbon or a carbon compound, theinsulation layer preferably comprising a silicon compound or a polymerlayer.

In one embodiment, it is preferred if the nanopores make up at leastapprox. 5%, preferably more than 60%, of the surface area of the firstelectrode.

In a further configuration of the pair of measuring electrodes accordingto the invention, only one or a few nanopores are provided per pair ofmeasuring electrodes. This configuration relates to an application inwhich redox processes at individual molecules are to be detected bymeasuring fluctuations in the current signal in order to obtaininformation as to the diffusion and kinetics of the redox process. Forthis purpose, it is necessary that only one or a very small number ofnanopores contributing to the overall current are lying in the electrodestructure, which in terms of its external dimensions and in view of thecost aspect, may still be on a micrometer scale. In this configuration,the amplitude of fluctuations relative to the mean of the measurementcurrent then becomes so great that it can be measured. Therefore, forcertain applications it is preferred and advantageous if only one or asmall number of nanopores are present in the pair of measuringelectrodes. The number of nanopores can easily be set during theproduction process, which is described in more detail below.

In this configuration, it is possible to use the novel pair of measuringelectrodes to measure current fluctuations in order to clarify diffusionand kinetics of redox molecules.

Another object of the present invention relates to a biosensor at leastcomprising one pair of measuring electrodes of this type, and to anelectrochemical cell comprising a biosensor of this type.

The biosensor may include a multiplicity of the novel pairs of measuringelectrodes, which can all be read out by dedicated supply lines. Thepairs of measuring electrodes are preferably interrogated or read-outvia a potentiostat circuit, the reference electrode and counterelectrodeof which may be arranged either in an electrochemical cell in which thebiosensor is located and to which an electrolyte containing molecules tobe measured is added, or on the biosensor itself.

In general terms, it is preferred if at least one further electrode,which serves as a reference electrode or counterelectrode and preferablyhas a surface area which is greater, preferably at least 10 timesgreater, than the surface area of the second electrode, is provided onthe substrate in the biosensor. In one embodiment, the biosensor isdesigned as a chip with supply conductors for the electrodes.

In one embodiment, the electrochemical cell has a receiving space for anelectrolyte in which molecules which are to be recorded using thebiosensor are present, the electrochemical cell preferably havingterminals for a readout circuit, preferably a potentiostat circuit.

The specificity for redox molecules which are to be analyzed can be setby coating the pores and/or electrodes. In this case, it is possible touse monolayers, polymer layers, in particular polyelectrolyte layers andhydrogels. The electropolymerization process is particularly preferredin this context, since localization of the coating specifically on theelectrodes and in the nanopores can be induced automatically by theapplication of current. The nanopores can in this case be coated with anion-selective membrane or with a membrane with embedded enzymes. For asensor application, it is also possible to provide a coating withpolymer and an embedding of redox-active molecules byelectropolymerization.

The novel pair of measuring electrodes can also be used as a microsensorfor microelectrode arrangements in order to detect neurotransmitters andnitrogen oxides (NO). The production of the pairs of measuringelectrodes can be easily integrated in the process for producingmicroelectrode arrangements. This results in the advantage of highsensitivity on account of the nanopores, also exploiting the advantagethat a physiological signal can be measured in the form of theconcentration of chemical substances.

The novel pairs of measuring electrodes can in principle be produced invarious ways. It is possible to use the following process, whichlikewise is an object of the invention:

a) applying a first, preferably sheet-like electrode to an insulatingsubstrate, the electrode preferably having a layer thickness of approx.50 to approx. 1000 nm, particularly preferably of approx. 100 to 200 nm,

b) applying an insulation layer to the first electrode,

c) masking the insulation layer using a nanostructured shadow mask madefrom nanoparticles which preferably have a diameter of 20 to 1000 nm,particularly preferably of approx. 100 nm,

d) applying a second electrode to the insulation layer, without anyelectrode material being deposited in the region of the nanoparticles,the electrode having a layer thickness in the region of the radius ofthe nanoparticles, preferably a layer thickness of approx. 20 to approx.500 nm, particularly preferably of approx. 50 nm,

e) removing the nanoparticles, and

f) etching the insulation layer up to the first electrode, with thesecond electrode serving as an etching mask.

An advantage of this process is that there is no need forphotolithography or electron beam lithography to be used to structurethe nanopores, and the process is very simple and inexpensive to carryout. The novel process makes it possible to produce extremely smallinter-electrode distances in a simple way and to achieve acorrespondingly high efficiency of the redox recycling, i.e. a highsensitivity.

In the novel process, therefore, first of all a first, sheet-likeelectrode is applied to a substrate, and then an insulation layer isapplied to the first electrode. Conventional photolithography can beused to microstructure the contours of the first electrode. Then, astructured shadow mask made from nanoparticles is deposited on theinsulation layer before the second, sheet-like electrode is applied.After the nanoparticles have been removed, a nanostructured upperelectrode remains, which is then used as an etching mask in order toetch the insulation layer selectively in the region of the openings inthe upper electrode. The etching process stops at the lower electrode,so that nanopores are formed, in which the lower electrode is partiallyexposed.

The nanoparticles are preferably deposited from a solution where theyare deposited regularly, in a random distribution, by means of aself-organization process.

The density of the nanoparticles acting as shadow masks on theinsulation layer can be set by means of the concentration ofnanoparticles in the solution. This is because on the one hand it may beadvantageous for sensor applications to produce a density of nanoporesas high as possible in order to achieve a high current density. On theother hand, to observe redox processes at individual molecules, it maybe advantageous to produce a sensor electrode having only one or a smallnumber of nanopores.

A process for producing nanostructured electrodes for measurements atimmobilized biomolecules is known from the publications by Musil et al.,J. Vac. Sci. Technol. 13 (1995), 2781-2786, and also Padeste et al., J.Electrochem. Soc. 143 (1996), 3890-3895.

In the known process, gold electrodes are deposited on an insulatingsubstrate which, as shadow mask, includes randomly distributednanoparticles. After the nanoparticles have been removed, what remainsis a gold electrode with irregularly arranged holes in which thesubstrate is uncovered. Analyte molecules, such as for exampleantibodies, are immobilized on the substrate in the holes and can inthis way be arranged very close to the readout electrode. Neitherpublication deals with pairs of measuring electrodes or with redoxrecycling.

To produce the novel pair of measuring electrodes, it is also possibleto employ the following process, which also is an object of theinvention:

a) applying a first, preferably sheet-like electrode to an insulatingsubstrate, the electrode preferably having a layer thickness of approx.50 to approx. 1000 nm, particularly preferably of approx. 100 to 200 nm,

b) masking the first electrode using a nanostructured shadow mask madefrom nanoparticles, which preferably have a diameter of 10 to 1000 nm,particularly preferably of approx. 100 nm,

c) applying an insulation layer to the first electrode, without anyinsulation material being deposited in the region of the nanoparticles,

d) applying a second electrode to the insulation layer, without anyelectrode material being deposited in the region of the nanoparticles,the electrode having a layer thickness in the region of the radius ofthe nanoparticles, preferably a layer thickness of approx. 20 to approx.500 nm, particularly preferably of approx. 50 nm, and

e) removing the nanoparticles.

As an alternative to the above-described applying of the nanoparticlesto the insulation layer as shadow mask for the electrode deposition, inthis case the nanoparticles have already been applied to the firstelectrode. Then, insulation layer and second electrode are deposited andthe nanoparticles are dissolved again. As a result, the insulation layerand the second electrode are structured directly, and the nanopores havethen been completed, eliminating the etching process from step f) of theother process.

The nanostructured shadow mask used may either be nanoparticlesdeposited from a solution or alternatively a shadow mask produced bymicrotechnology, i.e. a grid with nanopores through which vapordeposition or sputtering is carried out, in which case subsequentinversion of the structure of islands produced in this way can beeffected by known thin-film and micropatterning processes. Thenanostructuring may also be carried out by applying a mask using astamping process (nanoprinting) or by a forming process using ananostructured stamp (nanoimprinting).

The advantage in this case is that shadow mask or stamp only has to beproduced once using high-resolution lithography methods, then afterwardscan be used to manufacture a large number of sensors. Since it is onlythe size of the nanopores rather than their absolute position on theelectrode which is important, there is no need for any particularlyaccurate alignment of the tool, making the production processcorrespondingly inexpensive.

According to another object, the production process comprises the stepsof:

a) applying a first, preferably sheet-like electrode to an insulatingsubstrate, the electrode preferably having a layer thickness of approx.50 to approx. 1000 nm, particularly preferably of approx. 100 to 200 nm,

b) applying an insulation layer to the first electrode,

c) applying a nanostructured second electrode to the insulation layer,and

d) etching the insulation layer as far as the first electrode, with thesecond electrode being used as an etching mask.

As an alternative to a nanostructured shadow mask for producing thesecond electrode or for use as an etching mask, it is also possible toproduce a nanostructured second electrode by evaporation coating orsputtering or PECVD (plasma-enhanced chemical vapor deposition). Ifknown low-melting materials, such as for example gold, tin, silver,indium, are used, at layer thicknesses of above approximately 5-10 nm alayer which does not yet effect complete coverage but is laterallyconductive is produced. This process initially forms islands which growtogether as the layer thickness increases. A layer of this type islaterally conductive within a certain layer thickness range, typicallyapprox. 10 to 100 nm, but not completely continuous. A nanostructuredlayer of this type, like the layers produced with the aid ofnanoparticles or nanostructured shadow masks, can be used both as anetching mask and as a second electrode. In this context, it isadvantageous that there is no dedicated structuring step and there is noneed to apply nanoparticles or other nanostructured shadow masks.

This production of a nanostructured shadow mask can take place in thefollowing way:

Gold is vaporized in vacuo from a graphite crucible and is deposited ona suitable substrate. The coating rate is set at approximately 0.2-0.5nm/s. The substrate used is, for example, a film of amorphous carbon, asis customary for layer analysis in transmission electron microscopy. Thesubstrate temperature is 30° C. Under these conditions, first of allisolated nuclei are formed, and as the layer thickness increases thesenuclei grow together, forming a laterally conductive but nanoporouslayer.

The principles of nucleation and layer growth, which form the basis ofthis process, are extensively described in: Constantine A. Neugebauer:Condensation, Nucleation, and Growth of Thin Films, in: Handbook of ThinFilm Technology, Leon I. Maissel and Reinhard Glang (eds), ch. 8, p. 32ff. McGraw-Hill, 1970.

Advantageous configurations of the novel process and of the novel pairof measuring electrodes, of the biosensor and of the electrochemicalcell are given in the dependent claims.

It will be understood that the abovementioned features can be used notonly in the combination given but also on their own or in othercombinations without departing from the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Further advantages and features will emerge from the description and theappended drawing, in which:

FIG. 1 shows an electrochemical cell in which a biosensor comprising apair of measuring electrodes is illustrated in cross section and inexcerpt form, not to scale, with exaggerated dimensions;

FIG. 2 shows a plan view of the biosensor from FIG. 1; and

FIG. 3 shows a potentiostat circuit for reading out the electrochemicalcell from FIG. 1.

DESCRIPTION OF CERTAIN INVENTIVE EMBODIMENTS

FIG. 1 shows a highly schematized, cross-sectional view of anelectrochemical cell 10, in which a biosensor 11 is arranged which isused to measure molecules in an electrolyte located in a receiving space13 of the cell 10. Terminals 14, which can be used for electricalconnection of the biosensor 11, are arranged on the outside of the cell10.

The biosensor 11, which is shown in an enlarged, not-to-scale form,comprises an insulating substrate 15 on which a pair 16 of measuringelectrodes comprising a lower, first electrode 17 and an upper, secondelectrode 18 is arranged. Between the electrodes 17, 18 there is aninsulation layer 19 which holds the electrodes spaced apart from oneanother. In the second electrode 18 and the insulation layer 19 thereare nanopores 21 which extend as far as the surface 22 of the firstelectrode 17 which is uncovered in the nanopores 22. The nanopores arein a regular random distribution.

A process for producing a pair of measuring electrodes 17, 18 of thistype comprises the following steps:

Applying a first, preferably sheet-like electrode 17 to an insulatingsubstrate 15 made from glass, silicon/silicon oxide or a polymer layer,by sputtering or vapor deposition of gold, platinum, palladium, iridiumor carbon, the electrode 17 preferably having a layer thickness ofapprox. 100 to 200 nm. In the case of carbon electrodes, a broaderpotential range is accessible than with metal electrodes, andconsequently carbon electrodes are preferred for certain applications.

Applying an insulation layer 19 by sputtering or vapor deposition ofsilicon oxide or by spinning on a thin polymer film onto the firstelectrode 17. The layer thickness is preferably approx. 50 nm.

One further option for applying electrode and insulation layers is PECVD(plasma-enhanced chemical vapor deposition).

Next, the surface of the insulation layer 19 is pretreated in order toachieve a uniform distribution of nanoparticles on the surface. Thepretreatment is dependent on the type of nanoparticles and the solventin which they are brought to the surface. The surface may, for example,be made hydrophobic or hydrophilic, so that it can be wetted moresuccessfully.

Masking the insulation layer 19 with a shadow mask made fromnanoparticles which have a diameter of approx. 100 nm and are present ina solution (water, ethanol, toluene).

As an alternative to a shadow mask made from nanoparticles, it is alsopossible to use vapor-deposition particles, i.e. clusters, which form ina protective gas atmosphere, or a mask, which is produced bynanoprinting, nanoimprinting or vapor deposition or sputtering through amask with nanopores produced by microtechnology.

Applying a second electrode 18 by sputtering or vapor deposition ofmetal on the insulation layer 19, without any electrode material beingdeposited in the region of the nanoparticles. The electrode 18 acquiresa layer thickness which is in the region of the radius of thenanoparticles, preferably of approx. 50 nm.

Removing the nanoparticles, for which purpose the electrode 18 isexposed to ultrasound in a solvent, so that the nanoparticles (forexample of latex) are removed from the surface.

Etching the insulation layer 19 as far as the first electrode 17, withthe second electrode 18 serving as an etching mask. Wet-etchingprocesses or dry-etching processes can be used for this purpose. In thisway, the nanopores 21 in which the surface 22 of the first electrode 17is uncovered are produced.

The processes of sputtering, vapor deposition and etching are known tothe person skilled in the art, and in this context reference is made tothe specialist literature. The masking of the insulation layer 19 maytake place in the same way as described by Musil et al., loc. cit., orby Padeste et al., loc. cit.

FIG. 2 shows the biosensor 11 from FIG. 1 in a plan view which is not toscale. It can be seen that further electrodes 23, 24, which serve asreference electrode and counterelectrode for a potentiostat circuit, arepresent on the substrate 15. The further electrodes 23, 24 have asurface area which is considerably larger than that of the secondelectrode 18. The nanopores 21 are in this case merely indicated as dotson the second electrode 18. Furthermore, on the substrate 15 supplylines 25, 26, 27 and 28 can be seen, which lead to the first electrode17, the second electrode 18 and the further electrodes 23, 24,respectively. These supply lines are connected to the terminals 14 ofthe cell 10 shown in FIG. 1 when the biosensor 11 is inserted into thecell 10.

The pair 16 of measuring electrodes illustrated in FIGS. 1 and 2 canalso be used to measure redox processes at individual molecules. In thiscase, only one or a small number of nanopores are produced in the secondelectrode 18 and in the insulation layer 19, while the contourdimensions of the electrodes 17 and 18, as in the embodiment with alarge number of nanopores, may be in the micrometer range. On account ofits external dimensions on the micrometer scale, the electrode structurecan then still be produced at low cost, but on account of the fact thatonly one nanopore or a small number of nanopores is present, it ispossible to detect measurements of fluctuations in the current signal inorder to obtain information as to diffusion and kinetics of the redoxprocess.

FIG. 3 shows the cell 10 from FIG. 1 connected to a potentiostat circuit31 for read-out of the biosensor 11. The reference electrode 23 and thecounterelectrode 24 may also be provided in the cell 10 instead of onthe biosensor 11. With regard to the function of the potentiostatcircuit 31, reference is made to the corresponding literature which isaccessible to the person skilled in the art.

The reference electrode 23 is connected to the inverting input of apotentiostat 32, the non-inverting input of which is at groundpotential. The output of the potentiostat 32 is connected to thecounterelectrode 24.

The supply line 26 leading to the second, upper electrode 18 of thebiosensor 11 is connected to the output of a first differentialamplifier 33 with negative feedback, with a series resistor 34 connectedbetween them. Set potential E2 is applied to the non-inverting input ofthe differential amplifier 33, while the inverting input is connected tothe supply line 26.

The voltage drop across the series resistor 34 is measured using adifferential amplifier 35, the output of which therefore supplies avoltage signal which is proportional to the current flowing across thesecond electrode 18.

Correspondingly, the supply line 25 is connected to an output of asecond differential amplifier 36 with negative feedback, likewise with aseries resistor 37 connected between them. Set potential E1 is appliedto the non-inverting input of the differential amplifier 36, while theinverting input is connected to the supply line 25 which leads to thefirst electrode 17 of the biosensor 11.

The voltage across the series resistor 37 is recorded using adifferential amplifier 38, which at its output supplies a voltage signalwhich is proportional to the current flowing across the first electrode17.

1. A pair of measuring electrodes, comprising a first and a secondelectrode, and an insulation layer arranged between the electrodes,wherein one or more nanopores are provided in the second electrode, andwherein the nanopores extend through the insulation layer to the firstelectrode, the surface of which is at least partially uncovered by thenanopores, wherein the nanopores have an opening width selected from therange of approximately 20 nm to approximately 1000 nm.
 2. The pair ofmeasuring electrodes according to claim 1, wherein the electrodes aresheet-like.
 3. The pair of measuring electrodes according to claim 1,wherein the nanopores are distributed regularly.
 4. The pair ofmeasuring electrodes according to claim 1, wherein the nanopores aredistributed randomly.
 5. The pair of measuring electrodes according toclaim 1, wherein the insulation layer has a thickness selected from therange of approximately 10 nm to approximately 200 nm.
 6. The pair ofmeasuring electrodes according to claim 1, wherein the insulation layerhas a thickness of approximately 50 nm.
 7. The pair of measuringelectrodes according to claim 1, wherein the electrodes have a diameterselected from the range of approximately 1 μm to approximately 10 mm. 8.The pair of measuring electrodes according to claim 1, wherein theelectrodes have a diameter of approximately 10 μm.
 9. The pair ofmeasuring electrodes according to claim 1, wherein the electrodes areapplied to an insulating substrate.
 10. The pair of measuring electrodesaccording to claim 9, wherein the insulating substrate comprises atleast one of glass, silicon/silicon oxide, and a polymer.
 11. The pairof measuring electrodes according to claim 1, wherein the electrodescontain metal.
 12. The pair of measuring electrodes according to claim11, wherein the electrodes contain at least one of gold, platinum,palladium, iridium, carbon, and a carbon compound.
 13. The pair ofmeasuring electrodes according to claim 1, wherein the insulation layerincludes a silicon compound or a polymer layer.
 14. The pair ofmeasuring electrodes according to claim 1, wherein the nanopores form aproportion of at least approximately 5% of the surface area of the firstelectrode.
 15. The pair of measuring electrodes according to claim 14,wherein the nanopores form a proportion of at least 60% of the surfacearea of the first electrode.
 16. An electrochemical cell comprising abiosensor, wherein the biosensor comprises at least one pair ofmeasuring electrodes, wherein the pair of measuring electrodes comprisesa first and a second electrode and an insulation layer arranged betweenthe electrodes, wherein one or more nanopores are provided in the secondelectrode, wherein the nanopores extend through the insulation layer tothe first electrode, the surface of which is at least partiallyuncovered by the nanopores, and wherein the nanopores have an openingwidth selected from the range of approximately 20 nm to approximately1000 nm, wherein the cell comprises a receiving space for anelectrolyte, and wherein the electrolyte includes molecules to berecorded using the biosensor.
 17. The electrochemical cell according toclaim 16, wherein the cell comprises terminals for a readout circuit.18. The electrochemical cell according to claim 17, wherein the readoutcircuit is a potentiostat circuit.
 19. The electrochemical cell of claim16, wherein the insulation layer has a thickness selected from the rangeof approximately 10 nm to approximately 200 nm.
 20. A method ofmanufacturing a pair of measuring electrodes, comprising: a) applying afirst electrode to an insulating substrate; b) applying an insulationlayer to the first electrode; c) masking the insulation layer using ananostructured shadow mask made from nanoparticles; d) applying a secondelectrode to the insulation layer, without any electrode material beingdeposited in the region of the nanoparticles, wherein the secondelectrode has a layer thickness in the region of the radius of thenanoparticles; e) removing the nanoparticles; and f) etching theinsulation layer as far as the first electrode, wherein the secondelectrode serves as an etching mask.
 21. The method according to claim20, wherein, prior to step c), the surface of the insulation layer ispretreated in order to achieve a uniform distribution of thenanoparticles on the surface.
 22. The method according to claim 20,wherein the first electrode has a layer thickness selected from therange of approximately 50 nm to approximately 1000 nm.
 23. The methodaccording to claim 20, wherein the nanoparticles have a diameterselected from the range of approximately 20 nm to approximately 1000 nm.24. The method according to claim 20, wherein the second electrode has athickness selected from the range of approximately 20 nm toapproximately 500 nm.
 25. A method of manufacturing a pair of measuringelectrodes, comprising: a) applying a first electrode to an insulatingsubstrate; b) masking the first electrode using a nanostructured shadowmask made from nanoparticles; c) applying an insulation layer to thefirst electrode, without any insulation material being deposited in theregion of the nanoparticles; d) applying a second electrode to theinsulation layer, without any electrode material being deposited in theregion of the nanoparticles, wherein the second electrode has a layerthickness in the region of the radius of the nanoparticles; and e)removing the nanoparticles.
 26. The method according to claim 25,wherein the first electrode has a layer thickness selected from therange of approximately 50 nm to approximately 1000 nm.
 27. The methodaccording to claim 25, wherein the nanoparticles have a diameterselected from the range of approximately 20 nm to approximately 1000 nm.28. The method according to claim 25, wherein the second electrode has alayer thickness selected from the range of approximately 20 nm toapproximately 500 nm.
 29. A method of manufacturing a pair of measuringelectrodes, comprising: a) applying a first electrode to an insulatingsubstrate; b) applying an insulation layer to the first electrode; c)applying a nanostructured second electrode to the insulation layer; andd) etching the insulation layer as far as the first electrode, whereinthe second electrode serves as an etching mask.
 30. The method accordingto claim 29, wherein the layer thickness of the first electrode isselected from the range of approximately 50 nm to approximately 1000 nm.31. The method according to claim 29, wherein the layer thickness of thefirst electrode is selected from the range of approximately 100 nm toapproximately 200 nm.
 32. A method of manufacturing a pair of measuringelectrodes, comprising: a) applying a first electrode to an insulatingsubstrate; b) applying an insulating layer to the first electrode; c)applying a second electrode to the insulating layer; and d) producingnanopores in the second electrode, extending through the insulationlayer to the first electrode, wherein the surface of the first electrodeis at least partially uncovered by the nanopores.
 33. The methodaccording to claim 32, wherein the layer thickness of the firstelectrode is selected from the range of approximately 50 nm toapproximately 1000 nm.